The present invention relates to a method for producing an antibacterial agent, especially, indolmycin useful as an anti-H.pylori (Helicobacter pylori), by a fermentation.
Indolmycin is a compound represented by the following formula and useful as a pharmaceutical (for example, an antibacterial agent, especially anti-H.pylori agent), a veterinary agent and a herbicide. 
A known method for producing indolmycin by a chemical synthesis is a method described in Journal of Organic Chemistry, Vol.51, p. 4920 (1986). However, this synthesis gives only a racemate since indolmycin contains 2 asymmetric carbon atoms. While another method is described in Chemistry Letters, p.163 (1980), it involves a complicated process for obtaining an optically active indolmycin and has a low yield.
Still another known method for producing indolmycin is a fermentation using Streptomyces griseus ATCC 12648 as a producer microorganism (The Journal of Antibiotics, Vol.27, p.49 (1974), The Journal of Antibiotics, Vol.34, p.551 (1981)), but its low yield is also problematic from an industrial point of view.
Since a conventional method for producing indolmycin cannot satisfactorily be applied to industrial production, a large scale and convenient method for producing indolmycin is desired.
We made an effort under the circumstance described above to establish a method for producing indolmycin by fermentation, and finally discovered that by obtaining a tryptophan analogue-resistant mutant of Streptomyces griseus and by using it, an accumulation of a large amount of indolmycin in a culture broth can be obtained. We also discovered that by supplementing the medium with L-tryptophan a further higher accumulation can be achieved. We also established a method for obtaining indolmycin conveniently from the culture broth at a higher yield by means of chromatography using an adsorption resin, and a weakly-basic anion exchange resin, a weakly acidic cation exchange resin as a support together with an aqueous organic solvent.
Thus, the present invention relates to:
(1) A microorganism having indolmycin-producing ability and tryptophan analogue resistance;
(2) A microorganism according to the above (1) wherein said microorganism is a microorganism belonging to the Genus Streptomyces;
(3) A microorganism according to the above (1) wherein said microorganism is a microorganism belonging to the species Streptomyces griseus; 
(4) A microorganism according to the above (1) wherein said tryptophan analogue is fluoro-DL-tryptophan;
(5) A method for producing indolmycin or its salt comprising cultivating a microorganism according to the above (1) in a medium to allow indolmycin or its salt to be accumulated in the culture broth, and then recovering a product;
(6) A method according to the above (5) wherein L-tryptophan is added to the medium;
(7) A method according to the above (5) wherein 0.2 to 5 g of L-tryptophan is added per 1 L of the medium;
(8) A method according to the above (5) wherein 0.2 to 10 g of anthranilic acid is added per 1 L of the medium;
(9) A method according to the above (5) wherein 0.2 to 3 g of L-tryptophan and 0.2 to 2 g of anthranilic acid are added per 1 L of the medium;
(10) A method according to the above (5) wherein a purification is performed using an adsorption resin, a basic anion exchange resin and/or an acidic cation exchange resin as a support and an aqueous organic solvent as an eluent.
A tryptophan analogue employed in the invention may for example be a halogenated tryptophan such as 5-fluoro-DL-tryptophan (hereinafter sometimes referred to as 5-FT), 6-fluoro-DL-tryptophan (hereinafter sometimes referred to as 6-FT) and the like. One preferable example is 5-fluoro-DL-tryptophan or 6-fluoro-DL-tryptophan.
A microorganism having tryptophan analogue resistance can be obtained by subjecting a microorganism having an indolmycin-producing ability to a mutation-inducing treatment ordinarily employed in manipulating microorganisms followed by screening for a strain capable of growing in a tryptophan analogue-containing medium described above. Such mutation-inducing treatment may for example be (1) a UV irradiation [spores of a microorganism having an indolmycin-producing ability as a parent strain are suspended for example in a 100 mM phosphate buffer (pH7.0) and irradiated for 90 seconds under a 15 W UV lamp placed at a distance of 30 cm] and (2) a chemical treatment for example with N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine (hereinafter sometimes referred to as NTG) [NTG treatment: spores of a microorganism having an indolmycin-producing ability as a parent strain are suspended for example in a 50 mM Tris.HCl buffer (pH8.0) supplemented with 1 mg/ml of NTG and allowed to stand at 30xc2x0 C. for 1 hour].
Instead of a mutation-inducing treatment, a spontaneous mutation may also be employed to allow a strain to acquire ability of growing in a tryptophan analogue-containing medium, and such strain is encompassed in a microorganism having a tryptophan analogue resistance according to the invention.
A microorganism having an indolmycin-producing ability employed in this invention may be any microorganism having an indolmycin-producing ability, such as a microorganism of Streptomyces (for example, Streptomyces griseus), typically, Streptomyces griseus ATCC 12648 and Streptomyces sp. HC-21(IFO 15984, FERM BP-5571). The microbiological characteristics of Streptomyces sp. HC-21 are found in WO97/49703.
A microorganism having an indolmycin-producing ability and a tryptophan analogue resistance can be obtained as a tryptophan analogue-resistant mutant for example by subjecting an indolmycin-producing microorganism such as Streptomyces griseus ATCC 12648 as a parent strain to a mutation-inducing treatment such as a UV irradiation and an NTG treatment followed by incubating the strain in a medium (such as agar plate) containing a tryptophan analogue described above at a concentration at which the parent strain can not grow followed by screening the resultant colonies. The degree of the resistance of a mutant thus obtained against a tryptophan analogue can be determined for example as follows. Thus, an agar plate, for example as shown in Table 1, is supplemented with a certain amount of a tryptophan analogue (such as 5-fluoro-DL-tryptophan) and inoculated with a loopful of spores containing about 1xc3x97106 to about 1xc3x97108 spores/ml and then incubated under a condition suitable for the growth of the parent strain (for example at 24xc2x0 C. for 8 days), whereby determining the concentration of tryptophan analog at which the parent strain can grow. xe2x80x9cA microorganism having a tryptophan analogue resistance (a tryptophan analogue-resistant strain)xe2x80x9d according to the invention is a mutant which became to be able to grow in a medium (such as agar medium) containing a tryptophan analogue at a concentration at which the parent strain can not grow.
A microorganism having a tryptophan analogue resistance according to the invention may be a microorganism which can grow in a medium (such as agar plate) containing a tryptophan analogue described above at a high concentration. Against 5-fluoro-DL-tryptophan, for example, a microorganism capable of growing at a concentration of 0.1 to 10000 xcexcg/ml, preferably 1 to 5000 xcexcg/ml, more preferably 10 to 2000 xcexcg/ml is employed. Against 6-fluoro-DL-tryptophan, a microorganism capable of growing at a concentration of 50 to 1000 xcexcg/ml, preferably 100 to 800 xcexcg/ml is employed.
By cultivating a microorganism having a tryptophan analogue resistance obtained as described above followed by quantifying indolmycin in the culture broth, a strain giving an increased indolmycin accumulation can be selected.
Examples of a microorganism having an indolmycin-producing ability and a tryptophan analogue resistance are those described in the following Examples including Streptomyces griseus 5FW-1-226-9 capable of growing in the presence of 5-FT at 10 xcexcg/ml, Streptomyces griseus 5FW-2-8-7 capable of growing in the presence of 5-FT at 2 mg/ml and Streptomyces griseus 6FW-1-8-5 capable of growing in the presence of 6FT at 0.5 mg/ml.
Streptomyces griseus 5FW-1-226-9 described above was deposited at the Institute for Fermentation (IFO) on Mar. 27, 1998 under the deposition No. IFO 16170 and also at the Industrial Science and Technology of the Ministry of International Trade and Industry (NIBH, 1-1-3, Higashi, Tsukuba, Ibaragi, Japan) on Apr. 30, 1998 under the deposition number FERM BP-6336.
Streptomyces griseus 5FW-2-8-7 was deposited at the IFO on Mar. 27, 1998 under the deposition No. IFO 16171 and also at the NIBH on Apr. 30, 1998 under the deposition number FERM BP-6337.
Streptomyces griseus 6FW-1-8-5 was deposited at the IFO on May 26, 1998 under the deposition No. IFO 16178 and also at the NIBH on Jun. 1, 1998 under the deposition number FERM BP-6381.
Streptomyces sp. HC-21 was deposited at the IFO on Jun. 12, 1996 under the deposition No. IFO 15984 and also at the NIBH on Jun. 25, 1996 under the deposition number FERM BP-5571.
A production method of the invention comprises cultivating a microorganism having an indolmycin-producing ability and a tryptophan analogue resistance to allow indolmycin or its salt to be produced and accumulated in a culture broth and then recovering a product.
In this method, a microorganism can be cultured by any ordinary culture method such as cultivation with or without shaking, cultivation with aeration and agitation (for example batch culture or fed-batch culture). For a large scale fermentation, cultivation with aeration and agitation is preferred.
For the purpose of maintaining the dissolved oxygen in a culture at a level suitable to the growth of a microorganism and also to the production of indolmycin by said microorganism, an incubation with aeration and agitation for supplying oxygen as desired may also be employed.
While the medium employed in such incubation may be liquid or solid as long as it contains nutrients which allow the growth of the microorganism employed, a large scale fermentation preferably employs a liquid medium.
Such medium may contain any assimilable carbon sources, digestive nitrogen sources, inorganic salts, trace nutrients and the like as desired. A carbon source may for example be glucose, lactose, sucrose, maltose, dextrin, starch, mannitol, sorbitol, glycerol, fats (e.g., soybean oil, olive oil, rice bran oil, sesame oil, lard, chicken oil), various fatty acids (e.g., lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid) and the like. A nitrogen source may for example be meat extract, yeast extract, dried yeast, soybean flour, soybean meal, corn steep liquor, polypeptone, peptone, cottonseed meal, molasses, urea, thiourea, ammonia, ammonium salts (e.g., ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium acetate) and the like. An inorganic salt may for example be sulfate, hydrochloride, carbonate, nitrate, phosphate, acetate and borate of sodium, potassium, copper, calcium, magnesium, iron, manganese, zinc, cobalt, nickel and the like. A trace nutrient may for example be amino acids (e.g., glutamic acid, aspartic acid, alanine, lysine, valine, methionine, proline), peptides (e.g., dipeptide, tripeptide), vitamins (e.g., Vitamin B1, Vitamin B2, nicotinic acid, Vitamin B12, Vitamin C and derivatives thereof), nucleic acids (e.g., purine nucleotide, pyrimidine nucleotide and derivatives thereof), which may be added for the purpose of promoting the microbial growth or increasing the product yield.
Otherwise, for the purpose of adjusting the pH of the medium, an inorganic or organic acid or alkali may also be added, or an antifoam agent such as a surfactant (for example Silicon KM70 (xe2x80x9cSHINETSU KAGAKUxe2x80x9d) or xe2x80x9cACTCOLxe2x80x9d 31-56 (xe2x80x9cTAKEDA CHEMICAL INDUSTRIESxe2x80x9d) may be added in an amount by which the microbial growth is not adversely affected.
The pH of the medium is about 4 to 9, preferably about 5 to 8. While the medium may previously be supplemented with a phosphate buffer or potassium carbonate in order to keep the pH within the range specified above, it is supplemented with an alkali hydroxide, aqueous ammonia or an ammonia gas when the pH becomes lower than a desired level or with a mineral acid such as hydrochloric acid or sulfuric acid or an organic acid such as acetic acid or citric acid when the pH becomes higher than a desired level.
When a microorganism having an indolmycin-producing ability and a tryptophan analogue resistance according to the invention is cultivated, L-tryptophan is added to a medium at 0.01 to 1% (0.1 to 10 g per 1 L), preferably at 0.02 to 0.5% (0.2 to 5 g per 1 L), more preferably at 0.02 to 0.3% (0.2 to 3 g per 1 L) for the purpose of increasing the indolmycin production. For the same purpose, anthranilic acid may also be added. In such case, anthranilic acid is added to a medium at 0.01 to 6% (0.1 to 60 g per 1 L), preferably at 0.02 to 1% (0.2 to 10 g per 1 L), more preferably at 0.02 to 0.2% (0.2 to 2 g per 1 L).
As a matter of course, each of L-tryptophan and anthranilic acid may be added alone or in combination with each other to the medium.
While anthranilic acid is less expensive when compared with L-tryptophan, it may sometimes be inhibitory on the growth of a producing microorganism. Accordingly, it may give a successful result when combined with L-tryptophan. In such case, the concentration of each component may be adjusted to achieve an acceptable expenditure, less growth inhibition and maximum indolmycin production. When both of L-tryptophan and anthranilic acid are added, each may be added in an amount smaller than that when added alone, and 1 L of a medium may be supplemented with L-tryptophan at 0.01 to 0.5% (0.1 to 5 g), preferably 0.02 to 0.3% (0.2 to 3 g) and with anthranilic acid at 0.02 to 1% (0.2 to 10 g), preferably 0.02 to 0.2% (0.2 to 2 g).
L-Tryptophan may be added to the medium at once at the initiation of cultivation or intermittently during cultivation. Anthranilic acid is added intermittently at the initiation of and/or during cultivation so that microbial growth is not adversely affected, whereby obtaining a successful result.
The culture temperature is a temperature which is suitable for the growth of a microorganism employed and also for the extracellular accumulation of indolmycin (for example, about 10 to 40xc2x0 C., such as about 22 to 30xc2x0 C. for Streptomyces griseus and a mutant thereof and about 18 to 28xc2x0 C. for Streptomyces sp. HC-21 and a mutant thereof).
While the culture time may be appropriately selected based on the culture condition, the cultivation may be continued until the accumulated indolmycin per unit volume of culture broth reaches maximal production, for example, over a period usually of about 2 days to 2 weeks.
In a method of the invention, indolmycin is contained mainly in the culture supernatant. Accordingly, the culture broth after completing the cultivation is subjected to a separation method known per se (e.g., centrifugation, filter pressing, ultrafiltration, ceramic filtration and other forcible filtration) to remove the cells, and the resultant filtrate is subjected to a separation and purification to yield indolmycin. In such procedure, an aggregation agent may previously be added or a heat treatment may be employed as desired. In order to recover indolmycin as being purified from a filtrate or supernatant obtained as described above, a known purification method ordinarily employed for purifying an fat-soluble substance, such as extraction with an organic solvent immiscible with water (for example ethyl acetate, isobutanol, methylisobutylketone and the like) followed by concentration, may be employed.
In the invention, a method described below, wherein a purification is performed using an adsorption resin, a basic anion exchange resin and/or an acidic cation exchange resin as a support and an aqueous organic solvent as an eluent, is employed to achieve a high yield production of indolmycin at a low cost.
Thus, a resultant filtrate is first loaded on an adsorption resin [e.g., xe2x80x9cDIAIONxe2x80x9d HP-20, xe2x80x9cSEPABEADSxe2x80x9d SP-207 or SP-850 (xe2x80x9cMITSUBISHI KAGAKUxe2x80x9d), XAD-2 (Rohm and Haas, USA), preferably xe2x80x9cSEPABEADSxe2x80x9d SP-850] at pH 5 to 9 to allow indolmycin to be adsorbed. The resin is then washed with an alkaline solution at a concentration of 0.01 to 1 M (e.g., sodium hydroxide, potassium hydroxide, sodium carbonate, ammonia and the like) and an acid at a concentration of 0.01 to 1 M (e.g., hydrochloric acid, sulfuric acid, phosphoric acid). In this procedure, the order of the alkaline and acid washing steps may be inverted, and the steps may be repetitively performed. Subsequently, the resin is washed further with 5 to 30% aqueous alcohol (e.g., methanol, ethanol, isopropyl alcohol) and indolmycin is then eluted with 20 to 60% aqueous alcohol. By completing the washing and elution steps with the aqueous alcohols at a temperature of about 30 to 50xc2x0 C., the volume of an eluent can be reduced.
The eluent thus obtained is then loaded on a basic anion exchange resin and/or an acidic cation exchange resin as a support to continue the purification.
Such basic anion exchange resin may for example be xe2x80x9cDIAIONxe2x80x9d PA-412, PA-306, SA21A or WA-30 (xe2x80x9cMITSUBISHI KAGAKUxe2x80x9d), Amberlite IRA-402 (Rohm and Haas, USA), Dowex 1 (Dow Chemical, USA), as well as a weakly basic anion exchange resin such as Amberlite IRA-67 (Rohm and Haas, USA), with a weakly basic anion exchange resin being particularly preferred.
A cation exchange resin described above may for example be Amberlite IR-120B, IR-200C (Rohm and Haas, USA), Dowex 50 W (Dow Chemical, USA), xe2x80x9cDIAIONxe2x80x9d PK-216 (xe2x80x9cMITSUBISHI KAGAKUxe2x80x9d), as well as a weaklyxe2x80x94acidic cation exchange resin such as Amberlite IRC-50 (Rohm and Haas, USA), xe2x80x9cLEWATITxe2x80x9d CNP-80 (Bayer, Germany), with a weakly-acidic cation exchange resin being particularly preferred.
In addition to the loading on a resin described above, an activated charcoal treatment (e.g., activated charcoal for chromatography (TAKEDA CHEMICAL INDUSTRIES) may be employed alone, in combination or sequentially, and the resin is washed preferably with an aqueous alcohol described above.
The resultant effluent and washings are combined and concentrated under reduced pressure to yield indolmycin as a crystal. The mixture to be concentrated under reduced pressure may be combined with an alcohol (e.g. methanol, ethanol, isopropyl alcohol) in an amount of about 20 to 50% based on the mixture to be concentrated under reduced pressure, whereby increasing the resultant crystal content. Recrystallization from aqueous alcohol (30 to 60% methanol or ethanol) may be performed, if necessary, to yield a further highly purified crystal.
Indolmycin obtained in this invention is useful as a pharmaceutical (for example, an antibacterial agent, especially anti-H.pylori agent), a veterinary agent and a herbicide.